|
| Status |
Public on Dec 25, 2010 |
| Title |
iPSC 3.6_Undifferentiated_biological rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
Undifferentiated human induced pluripotent stem cells (iPSC)
|
| Organism |
Homo sapiens |
| Characteristics |
biological source: NHSK - Normal Human Dermal Keratinocyte
source: Generated at Cincinnati Children's Hospital Pluripotent Stem Cell Facility
|
| Treatment protocol |
To induce Definitive Endoderm (DE) differentiation, stem cell cultures were treated for three days as follows: D1- 100 ng/ml Recombinant Human Activin A (R&D Systems), 25ng/ml recombinant human Wnt3a (R&D Systems) and 0% dFBS (Fisher/Hyclone); D2-100ng/ml Activin A, 0.2% dFBS; D3- 100ng/ml Activin A, 2%dFCS. Basal media for treatment is RPMI + L-glut + Non-Essential Amino Acids (Invitrogen).
|
| Growth protocol |
Undifferentiated stem cells were maintained in feeder free conditions using hES qualified matrigel (BD Biosciences) and mTeSR media (Stemcell Technologies).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with TRIzol Reagent according to Invitrogen's product instruction.
|
| Label |
biotin
|
| Label protocol |
The Affymetrix Whole Transcript Sense Target Labeling Assay (Affymetrix) is used to create biotin-labeled sense-strand cDNA targets for hybridization to the Gene 1.0 ST Arrays. Starting material is 100-300 nanograms of total RNA.
|
| |
|
| Hybridization protocol |
2.5 micrograms of labeled cDNA in 80 microliters of hybridization cocktail was hybridized to the GeneChip Human Gene 1.0 ST Array for 18 hours at 45 degrees Celsius. GeneChips were washed and stained with R-Phycoerythrin Streptavidin (Molecular Probes) and goat-Anti-Streptavidin biotinylated antibody (Vector Laboratories) using the Fluidics Station 450 (Affymetrix) utilizing the fluidics protocol FS450-0007.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G and analyzed with Affymetrix Genechip Operating Software.
|
| Description |
Gene expression profile for pluripotent stem cells
|
| Data processing |
Data was processed by creating an Affymetrix Exon Expression experiment with advanced analysis workflow In GeneSpring GX 10.0.2. The Probe Summarization algorithm was RMA16 with a core transcript level and a baseline transformation set to the median of all samples.
|
| |
|
| Submission date |
Nov 23, 2010 |
| Last update date |
Jan 14, 2011 |
| Contact name |
James M Wells |
| Organization name |
Cincinnati Children's Hospital Medical Center
|
| Department |
Developmental Biology
|
| Lab |
R3469
|
| Street address |
3333 Burnet Ave.
|
| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE25557 |
Characterization of Definitive Endoderm formation from HESC and iPSC lines by Microarray analysis |
|
